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Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
34326 45018 2015 7 PDF Available
Title
Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis
Abstract

•The metabolic pathway of TNT catalyzed by Escherichia coli nitroreductases was elucidated.•The final product of TNT reduction by NfsA/NfsB was identified as dihydroxylamines.•NfsB mutants F123A and F124W dramatically enhanced the catalytic activity.•The two adjacent phenylalanines displayed different effects on the catalytic activity.

2,4,6-Trinitrotoluene (TNT), the most widely used explosive, causes serious health and environment problems and is recalcitrant to degradation. Herein, we investigated the metabolic pathway of TNT catalyzed by purified nitroreductases NfsA and NfsB from Escherichia coli, and enhanced the transformation activity by structure-based mutagenesis. The two nitroreductases play similar roles in the transformation of TNT, which is converted initially to 4-hydroxylamino-2,6-dinitrotoluene and then 2,4-dihydroxylamino-6-nitrotoluene without production of any amine products. Of the mutants constructed, F123A and F124W mutants dramatically enhanced the catalytic activity towards TNT and its derivatives, suggesting that these two adjacent phenylalanines displayed different effects on the catalytic activity. Substitutions with Ala or Asn at Phe124 greatly impaired the reduction activity against TNT, while the mutation of Phe to Trp resulted in a substantially reduced apparent Km and improved activity, implying the importance of aromatic residues at position 124 for substrate binding. In contrast, Phe at position 123 caused adverse effects on the activity, may be due to the steric constraint limiting movement of Phe124.

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Keywords
2,4,6-Trinitrotoluene; E. coli nitroreductases; Transformation pathway; Structure-based mutagenesis
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Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 50, Issue 5, May 2015, Pages 705–711
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us